Ribonucleic acid (RNA) plays an essential role in the translation of the genetic code to produce proteins necessary for cellular function, both in normal cells and neoplastic or diseased cells. In particular, RNA including transfer RNA, messenger RNA or messenger-like RNA, and ribosomal RNA carry and translate the genetic code to sites of protein production. Further, double-stranded RNA and species therefrom, including small inhibitory RNA or siRNA, and micro RNA or miRNA, play an important role in silencing genetic expression. Other RNA species are found within ribonucleoproteins. For example, telomerase RNA is a critical component of telomerase, an important ribonucleoprotein highly expressed in most cancers. The pathogenesis and regulation of cancer is thus dependent upon RNA-mediated translation and/or inhibitory control of specific genetic code, said genetic code often reflecting mutational events or other alterations within deoxyribonucleic acid (DNA), including epigentic alterations such as hypermethylation, microsatellite alterations, loss of heterozygosity, translocations, deletions, and point mutations. Further, other RNA species and their associated proteins, although not necessarily being directly involved in neoplastic pathogenesis or regulation, may provide recognizable characterization of neoplasia or disease by being inappropriately expressed or elevated. Such overexpression of RNA thus can delineate cancer or other disease. Recognition of the presence or overexpression of specific RNA, including both coding and non-coding RNA, can enable identification, detection, inference, monitoring, or evaluation of any neoplasm, whether benign, malignant, or premalignant, in humans and animals.
U.S. Pat. No. 6,329,179 B1, incorporated herein in its entirety, teaches that both tumor-associated and non-tumor associated RNA are detectable in plasma and serum. Total RNA, to be understood in the cancer patient to comprise both tumor-associated and non-tumor-associated RNA and further being heterogeneous RNA, can be extracted from plasma or serum, the RNA of interest or its cDNA is amplified qualitatively or quantitatively, and the amplified product of an RNA or cDNA species of interest detected. Subsequent art supports these teachings by demonstrating that extracellular RNA of various RNA species are detectable in bodily fluids, for example in co-owned U.S. Pat. No. 6,607,898; Kopreski et al., 1999, Clin. Cancer Res. 5: 1961-1965; Dasi et al., 2001, Lab. Investigation 81: 767-769; Hasselmann et al., 2001, Oncol. Rep. 8: 115-118; Ng et al., 2002, Clin. Chem. 48: 1212-1217; Chen et al., 2000, Clin. Cancer Res. 6: 3823-3826; Silva et al., 2001, Clin. Cancer Res. 7: 2821-2825; Silva et al., 2001, Oncol. Rep. 8: 693-696; Gal et al., 2001, Ann. NY Acad. Sci. 945: 192-194; Durie et al., 2000, Acta Oncol. 39: 789-796; Fleischhacker et al., 2001, Ann. NY Acad. Sci. 945: 179-188; Miura et al., 2003, Oncology 64: 430-434; Kopreski et al., 2001, Ann. NY Acad. Sci. 945: 172-178; and Wong et al., J. Clin. Pathol. 2004, 57: 766-768, said references incorporated herein in their entirety. Detection of tumor-associated RNA in plasma or serum thus provides a method for detecting, diagnosing, inferring, or monitoring cancer or premalignancy in a human or animal.
Neoplasia is characterized by varying degrees of invasiveness, metastatic potential, and resistance or responsiveness to particular therapies. Furthermore, these characteristics for a given neoplasia may change over time, for example by becoming progressively more malignant, invasive, metastatic, heterogeneous, undifferentiated, or treatment-resistant. Phenotypic changes often reflect underlying molecular changes. In particular, the relative ratio of particular RNA species, including coding and non-coding species, to each other, and/or to DNA, and/or to proteins can determine the characteristics of the neoplasia.
Comparative analysis of extracellular RNA species to each other, and/or to extracellular DNA, and/or to extracellular protein, would thus be useful as a method for detecting, diagnosing, inferring, characterizing, or monitoring cancer or premalignancy in a human or animal. Said comparative analysis further enables the selection and monitoring of treatment. To optimize the reliability and reproducibility of such comparative analysis, there is a need in the art for methods that maintain the stability of extracellular plasma RNA species and their concentrations and relative ratios within a plasma or serum or bodily fluid specimen over time. Although extracellular RNA is generally protected from the degrading effects of plasma RNase by being protected within phospholipid-encapsulated particles such as apoptotic bodies, a variable disruption of the particles or apoptotic bodies may occur over time or consequent to mechanical manipulations such as freeze-thawing or centrifugation, leading to variable rates of degradation of the extracellular RNA through its exposure to nucleases. The present invention provides methods to enhance the intra-specimen stability and reproducibility of the ratio between two or more RNA species in a plasma or serum or bodily fluid specimen, and kits thereof.
Thus, there is a need in the art for methods of comparing the amount or concentration or relative ratio of two or more plasma or serum RNA species or fragments thereof to permit diagnosis, detection, inference, evaluation, or monitoring of neoplastic disease in a human or animal. It is to be explicitly understood that said comparison of two or more RNA species may include comparison of non-mutated tumor RNA to tumor RNA; tumor RNA to non-mutated non-tumor RNA; coding RNA to coding RNA; coding RNA to non-coding RNA; and non-coding RNA to non-coding RNA; or any combination thereof. There is a need in the art for methods of enhancing the reliability and reproducibility of said comparisons.
Furthermore, there is a need for methods of comparing the amount or concentration or ratio of one or more extracellular RNA species to the amount or concentration of total RNA or extracellular DNA or protein present in the plasma, serum, or bodily fluid of a human or animal for the diagnosing, detecting, inferring, evaluating, or monitoring cancer and other neoplastic diseases in the human or animal.